Natural Variation in Small Molecule–Induced TIR-NB-LRRSignaling Induces Root Growth Arrest via EDS1- andPAD4-Complexed R Protein VICTR in Arabidopsis C W

Tae-Houn Kim,a,1 Hans-Henning Kunz,a Saikat Bhattacharjee,b Felix Hauser,a Jiyoung Park,a Cawas Engineer,a

Amy Liu,a Tracy Ha,a Jane E. Parker,c Walter Gassmann,b and Julian I. Schroedera,2

a Division of Biological Sciences, Section of Cell and Developmental Biology, University of California at San Diego, La Jolla, California92093-0116bDivision of Plant Sciences, Christopher S. Bond Life Sciences Center and Interdisciplinary Plant Group, University of Missouri,Columbia, Missouri 65211-7310cMax-Planck Institute for Plant Breeding Research, Department of Plant-Microbe Interactions, D-50829 Cologne, Germany

In a chemical genetics screen we identified the small-molecule [5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione(DFPM) that triggers rapid inhibition of early abscisic acid signal transduction via PHYTOALEXIN DEFICIENT4 (PAD4)- andENHANCED DISEASE SUSCEPTIBILITY1 (EDS1)-dependent immune signaling mechanisms. However, mechanisms upstreamof EDS1 and PAD4 in DFPM-mediated signaling remain unknown. Here, we report that DFPM generates an Arabidopsis thalianaaccession-specific root growth arrest in Columbia-0 (Col-0) plants. The genetic locus responsible for this natural variant, VICTR(VARIATION IN COMPOUND TRIGGERED ROOT growth response), encodes a TIR-NB-LRR (for Toll-Interleukin1 Receptor–nucleotide binding–Leucine-rich repeat) protein. Analyses of T-DNA insertion victr alleles showed that VICTR is necessary forDFPM-induced root growth arrest and inhibition of abscisic acid–induced stomatal closing. Transgenic expression of the Col-0VICTR allele in DFPM-insensitive Arabidopsis accessions recapitulated the DFPM-induced root growth arrest. EDS1 andPAD4, both central regulators of basal resistance and effector-triggered immunity, as well as HSP90 chaperones and theircochaperones RAR1 and SGT1B, are required for the DFPM-induced root growth arrest. Salicylic acid and jasmonic acidsignaling pathway components are dispensable. We further demonstrate that VICTR associates with EDS1 and PAD4 ina nuclear protein complex. These findings show a previously unexplored association between a TIR-NB-LRR protein and PAD4and identify functions of plant immune signaling components in the regulation of root meristematic zone-targeted growth arrest.

INTRODUCTION

Chemical genetics provides a powerful tool to address re-dundancy and network robustness in signal transduction(Schreiber, 2000; Armstrong et al., 2004; Zouhar et al., 2004;Park et al., 2009). In a recent screen of a 9600-compoundchemical library for inhibition of abscisic acid (ABA) signaling,a small molecule named [5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione (DFPM) was isolated that showedthe capacity to inhibit several ABA responses, including rapiddisruption of ABA-induced stomatal closing and ABA activationof guard cell anion channels (Kim et al., 2011). Detailed analysesrevealed that DFPM stimulates an effector-triggered immune

signaling pathway and thereby rapidly disrupts ABA signaltransduction (Kim et al., 2011).The central regulators of basal resistance and effector-triggered

immunity, EDS1 and PAD4, as well as the cochaperonesRAR1 (REQUIRED FOR Mla12 RESISTANCE) and SGT1b(SUPPRESSOR OF G-TWO ALLELE OF SKP1b) are required forDFPM-induced disruption of ABA signaling (Kim et al., 2011).Effector-triggered immunity relies on resistance (R) proteins assensors that indirectly or directly recognize specific pathogen-derived effector molecules and trigger downstream disease re-sistance responses (Glazebrook, 2005; Jones and Dangl, 2006).Whether specific R proteins are required for the small-moleculeDFPM-induced response remains unknown. Depending on theirN-terminal domain, R proteins are generally divided into twogroups, coiled-coil (CC)-nucleotide binding (NB)-leucine-richrepeat (LRR) and Toll-Interleukin1 Receptor (TIR)-NB-LRR(Meyers et al., 2003; Belkhadir et al., 2004; Chisholm et al., 2006;DeYoung and Innes, 2006; Shen and Schulze-Lefert, 2007;Caplan et al., 2008). The genome of Arabidopsis thaliana con-tains ;150 NB-LRR genes, and up to 600 genes are found inrice (Oryza sativa; Kadota et al., 2010). The presence of manypolymorphisms in NB-LRR genes has been proposed as a di-versification process against rapidly evolving pathogens (Tianet al., 2003; Guo et al., 2011). In fact, NB-LRR genes representthe most variable plant gene family (Weigel, 2012).

1 Current address: College of Natural Sciences, Duksung Women’sUniversity, 132-714 Seoul, Korea.2 Address correspondence to jischroeder@ucsd.edu.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Julian I. Schroeder(jischroeder@ucsd.edu).C Some figures in this article are displayed in color online but in black andwhite in the print edition.W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.112.107235

The Plant Cell, Vol. 24: 5177–5192, December 2012, www.plantcell.org ã 2012 American Society of Plant Biologists. All rights reserved.

Upon pathogen recognition, intramolecular structural changesof NB-LRR proteins induce ADP-ATP exchange in the NB do-main and activate downstream responses. Effector-triggeredactivation of TIR-NB-LRR proteins requires the function of EDS1and PAD4 to generate downstream disease resistance re-sponses, including salicylic acid–mediated signal transduction(Wiermer et al., 2005). Recently, it was found that EDS1 residesin some complexes with TIR-NB-LRR proteins (Bhattacharjeeet al., 2011; Heidrich et al., 2011).

Here, using a chemical genetics approach, we identifiednatural genetic variation in a primary root growth response in-duced by the small molecule DFPM. The Arabidopsis Columbia-0(Col-0) accession responds specifically to DFPM by generat-ing a primary root meristem arrest. Molecular identification of theresponsible genetic locus and its functional characterizationdemonstrate that a natural genetic variant in the TIR-NB-LRRgene VICTR (for VARIATION IN COMPOUND TRIGGEREDROOT growth response) determines DFPM-induced root growtharrest. Involvement of additional components of effector-triggered immune signaling in this phenotype implicates pathogen-induced primary root growth arrest as a possible resistanceresponse to soil-borne plant pathogens. Furthermore, we showan association of VICTR protein with EDS1 and with PAD4 withina nuclear complex. The finding of PAD4 within a TIR-NB-LRR–containing protein complex expands recent findings that TIR-

NB-LRR R proteins can associate within complexes with theEDS1 protein (Bhattacharjee et al., 2011; Heidrich et al., 2011).The DFPM-induced PAD4- and EDS1-dependent root growtharrest is caused by inhibition of primary root meristem activity.The DFPM-induced and easily scored root growth arrest pro-vides a powerful tool for further dissection of R protein/EDS1/PAD4-dependent signaling mechanisms, as genetic and cellbiological components of immunity-induced cell death pathwayscan be identified, as shown here for VICTR.

RESULTS

Accession-Specific Primary Root Growth Response to theSmall Compound DFPM

The chemical compound DFPM (Figure 1A) was isolated froma chemical library of 9600 compounds by screening for physi-ologically active small molecules that inhibit ABA-induced geneexpression and signal transduction (Kim et al., 2011). An in-depth screen for additional visible phenotypic responses causedby DFPM application identified a rapid root growth inhibitionafter DFPM treatment. Intriguingly, this arrest was found to bespecific for the Col-0 accession. DFPM did not affect rootgrowth of the Arabidopsis Landsberg erecta (Ler) accession(Figure 1B). At a concentration of 5 mM DFPM, root growth of

Figure 1. Natural Genetic Variation in Compound-Specific Root Growth Arrest Specifically Targets the Primary Root Meristem in Col-0.

(A) Chemical structure of root growth arrest-causing DFPM.(B) DFPM causes a primary root growth arrest phenotype in Col-0.(C) DFPM-induced root growth inhibition of Col-0 (EC50 ;1.5 mM). Error bars mean 6 SD.(D) While Col-0 roots are sensitive to DFPM, roots of Ler, Br-0 (Brunn-0), Ts-1 (Tossa de Mar-1), Kin-0 (Kindalville-0), Bay-0 (Bayreuth-0), C24, RLD(Rschew), WS (Wassilewskija), and 42 other Arabidopsis accessions (see Supplemental Table 1 online) produced no significant growth arrest responseto DFPM. The black horizontal bars mark the starting position of roots when DFPM was applied.(E) Microscopy examination of DFPM-treated roots reveals that while root differentiation is normal, meristematic and elongation zones of the primaryroot are reduced in size in response to DFPM in Col-0. Note that Ler produced normal root morphology in response to DFPM exposure (left).Meristematic and elongation zones are indicated by lines and arrows. Bars = 50 mm.(F) Deregulated DR5 promoter expression by DFPM indicates that auxin accumulation is abrogated in the root meristem after 4 d of DFPM treatment.Expression of the endodermal marker gene SCR is also altered by DFPM. Expression of the CyclinB1 promoter suggests that cell cycle activity in theDFPM-treated primary root tip is reduced. All marker lines tested here are in the Col-0 background.

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Col-0 was blocked (EC50 = 1.5 mM; Figure 1C). The seedlingroot growth of 50 other Arabidopsis accessions, including Ler,Kindalville-0 (Kin-0), Bayreuth-0 (Bay-0), and C24, were not af-fected by DFPM treatment (Figure 1D; see Supplemental Table 1online). We concluded that the Col-0 accession contains unusualnatural genetic variation that causes DFPM-induced root growtharrest. Based on the phenotype, we refer to this class of genes asVICTR. In response to DFPM, primary root growth of Col-0 wasabrogated, but secondary lateral root growth was not (Figure 1B).

Chemical compounds structurally similar to DFPM (seeSupplemental Figure 1A online) were analyzed to define criticalstructural motifs required for the triggering of the VICTR phe-notype. These analyses showed that modifications in thestructure of several DFPM-related compounds can reduce thepotency of the DFPM response. Although the slightly modifiedmolecules DFPM-1 and DFPM-2 exhibited biological activitysimilar to the original DFPM, a chlorine in the four positionseemed to be critical for eliciting the VICTR phenotype (seeSupplemental Figure 1 online). Loss of activity was also causedby modifications of side groups as shown in DFPM-3. WhetherDFPM or a breakdown product thereof is the active compoundremains to be determined. The root growth response caused byDFPM was found to be time dependent. Only 8 h of DFPMtreatment was sufficient to initiate the VICTR phenotype in anirreversible manner (see Supplemental Figure 2C online). Pri-mary root growth did not recover even 6 d after seedlings wereretransferred to standard growth media.

DFPM Targets the Primary Root Meristematic Zone in Col-0

Microscopy examination of the morphological changes of Col-0roots treated with DFPM revealed a large reduction of themeristematic and elongation zones, whereas root differentiation,such as vascular and lateral root formation, did not show anyobservable differences, indicating that DFPM may target theprimary root meristematic zone (Figure 1E). To investigate mo-lecular changes in DFPM-treated primary root tips, the pattern oflocal auxin accumulation was examined using DR5 promoter-driven green fluorescent protein (GFP) lines (Friml et al., 2003) inthe Col-0 background (Figure 1F). DFPM treatment for 1 d didnot change the pDR5:GFP activity at the columella at the root tip(see Supplemental Figure 2A online). After 4 d of DFPM treat-ment, no pDR5:GFP activity was visible at the columella of Col-0(Figure 1F), consistent with the model that DFPM targets theprimary root meristem. However, the pDR5:GFP construct hasbeen reported to have certain limitations as an auxin reporterand to not necessarily correlate with other auxin-sensitive genes(Moreno-Risueno et al., 2010). Therefore, to measure the effectof DFPM on auxin transporters in the primary root, pPIN1:PIN1-GFP and pPIN2:PIN2-GFP Col-0 lines were monitored asa function of time after DFPM exposure (see SupplementalFigure 2B online). After 18 h of DFPM treatment, both PIN1:GFPand PIN2:GFP signals became weaker. At 24 h, when DFPM-mediated root growth arrest becomes visible, both PIN1:GFPand PIN2:GFP levels decreased substantially. These results in-dicate that altered PIN1 or PIN2 activities and resulting changesin the auxin gradient, which is important to maintain meristematiccell divisions (Blilou et al., 2005; Dello Ioio et al., 2007; Fernández-

Marcos et al., 2011), might contribute to DFPM-mediated rootgrowth inhibition.SCARECROW (SCR) expression was analyzed as an endo-

dermal and quiescent center marker gene (Di Laurenzio et al.,1996). SCR expression was disrupted after 4 d of DFPM treat-ment when DFPM had already produced obvious morphologicaldifferences (Figure 1F). However, SCR expression exhibiteda normal pattern of expression after 1 d of DFPM treatment (seeSupplemental Figure 2A online). Therefore, the disrupted SCRexpression in Col-0 might be a secondary effect of the alteredroot morphology after DFPM treatment.Consistent with the deregulated DR5 and SCR expression

patterns induced after 4 d by DFPM, cell cycle activity monitoredby the Cyclin B1 promoter (Colón-Carmona et al., 1999) wasdisrupted by DFPM treatment (Figure 1F). Retention of the normalexpression pattern of Cyclin B1 in secondary root meristemsdemonstrates the tissue specificity of the DFPM effect (Figure1F). These marker line analyses suggesting that DFPM targets theprimary root meristematic zone led us to further explore howDFPM mediates root growth arrest (described further below).

The VICTR Gene Encodes a TIR-NB-LRR Protein

To identify the genetic element(s) in Col-0 producing the VICTRphenotype, 100 Col 3 Ler recombinant inbred lines (RILs)(Clarke et al., 1995) that have been genotyped using microarrays(Singer et al., 2006) were screened for the VICTR phenotype (seeSupplemental Table 2 online). Quantitative trait locus (QTL)analyses of the phenotypic scores and available genetic mapinformation for these RILs suggested that a single locus in Col-0positioned on the lower arm of chromosome 5 might determineDFPM-induced root arrest (Figure 2A).F1 seedlings of Col-0 crosses to Ler produced the Col-0

VICTR phenotype upon DFPM treatment, indicating that het-erozygous expression of the VICTRCol allele is sufficient toconfer the DFPM response in the Ler background. Consistentwith QTL analyses of the Col-0 3 Ler RILs, F2 seedlings ofa Col-0 cross to Ler segregated at a ratio of 3:1 (P value of x2

test is 0.665, n = 178), indicating that a single locus mediates theVICTR phenotype. Using these segregating VICTR F2 plants,map-based cloning narrowed down the candidate VICTR locusto a 100-kb region (Figures 2A and 2B). Since VICTR is a naturalvariant present in Col-0, any polymorphic gene within the targetregion could encode VICTR. Because VICTRCol was necessaryand sufficient for the DFPM response and was hypothesized torepresent an active allele, T-DNA insertion mutants of geneslocated within the target region in Col-0 were examined. Amongthe T-DNA lines tested, only T-DNA insertion lines in geneAt5g46520 displayed complete insensitivity in DFPM-mediatedroot growth arrest (Figures 2C and 2D). Five independent T-DNAinsertion mutants in the Col-0 At5g46520 gene showed loss ofDFPM-triggered root arrest.DFPM inhibits primary root growth in Col-0 but not in the victr

mutants. To determine how DFPM mediates this effect, confocalimages of Col-0, victr-1, and victr-2 primary roots were analyzed(see Supplemental Figure 3 online). While the quiescent centercells and cortical stem cells appeared unaffected by 24 h ofDFPM treatment in all three genotypes, the division zone of the

Natural Variation in TIR-NB-LRR Signaling 5179

DFPM-treated Col-0 primary roots was smaller than that of thevictr-1 and victr-2 primary roots. To further analyze this phe-notype, we quantified the number of meristematic cells in thedivision zones of primary roots (see Supplemental Figure 3 on-line). Our data indicate that when treated with DFPM for 24 h,Col-0 primary roots show a reduced number of meristem cells inthe division zone of the primary root compared with victr-1 andvictr-2. This shorter division zone and the reduced number ofmeristematic cells are consistent with the inhibition of primary

root growth first observable after 24 h of DFPM treatment in Col-0wild type and may represent the initial phase of DFPM-inducedgrowth inhibition.The most similar gene to VICTR, VICTR Like1 (VICTL1;

At5g46510), is located in tandem upstream of VICTR (Figure 2C;see Supplemental Figure 4B and Supplemental Data Set 1 on-line). A T-DNA mutant victl1-1 was not resistant to DFPMtreatment in the root growth arrest response (see SupplementalFigure 4C online). This suggests that the VICTRCol (At5g46520)

Figure 2. Identification of the Natural Variation VICTR Locus.

(A) QTL analyses using the 100 Col3 Ler RILs identified a single locus on chromosome 5 as a main element that triggers the VICTR phenotype in Col-0.lod, logarithm of odds.(B) Map-based cloning using F2 segregants of Col-0 crosses to Ler narrowed down the natural mutation VICTRCol within a 100-kb region.(C) Gene structure of VICTRCol and VICTL1 and the locations of T-DNA insertion mutants.(D) T-DNA insertion mutants of VICTRCol showed complete resistance to DFPM in root growth assays. The black horizontal bars mark the startingposition of roots when DFPM was applied. Error bars mean 6 SD (n = 3 with 10 plants each).(E) Protein motif structure of VICTR. Amino acid sequence comparison deduced from the cDNAs of Col-0, Bay-0, and Kin-0 identified multiple Col-0–specific amino acid polymorphisms within the coding region of VICTRCol. Vertical lines indicate positions where the Col-0 sequences differ from Bay-0and Kin-0. It is important to note that Bay-0 and Kin-0 sequences are identical in these positions. Protein sequences other than the TIR, NB, and LRRdomains are depicted by black horizontal bars. Asterisks mark amino acid deletions. Most amino acid polymorphisms are found in the NB domain (13polymorphisms/44 total) and the linker regions between the NB and LRR domains (24 polymorphisms/44 total).[See online article for color version of this figure.]

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gene is required for the chemical DFPM response targetingprimary root meristem activity. Chemical genetics can providean approach for identifying phenotypes in potentially redundantsignal transduction genes via small molecule targeting of spe-cific proteins (Park et al., 2009).

VICTR is predicted to encode a previously uncharacterizedmember of the NB-LRR family with a TIR motif (TIR-NB-LRR) atthe N terminus (Figure 2E). TIR-NB-LRR family members canfunction as immune signaling proteins encoded by R genes re-sponding to specific pathogen effectors in plants (Belkhadiret al., 2004; Chisholm et al., 2006; Jones and Dangl, 2006; Shenand Schulze-Lefert, 2007; Caplan et al., 2008). Compared withcharacterized R proteins, the predicted VICTRCol protein showshigh sequence similarity to that of At1g31540 (64.9% amino acididentity) (Katoh et al., 2002; Thompson et al., 2003; Raghava andBarton, 2006), the Col-0 allele of RAC1 from accession Ksk-1,which mediates PAD4-independent resistance to Albugo can-dida (Borhan et al., 2004). VICTRCol is in the same cluster of Rgenes as RPS6 (At5g46470; see Supplemental Figure 4B online)that specifies resistance to the Pseudomonas syringae effectorHopA1 (Kim et al., 2009).

To determine whether VICTRCol contains unique polymor-phism(s) that define the Col-0 sensitivity to DFPM, VICTRcDNA sequences from other accessions were reverse transcribedand compared with VICTRCol. The VICTR gene is highly divergedand possibly absent in Ler (Ziolkowski et al., 2009) andalso in Wassilewskija-0 (Ws-0) based on PCR analysis (seeSupplemental Figure 4E online). Comparison of the deducedamino acid sequences in the VICTR-containing ecotypes, Bay-0and Kin-0, shows that VICTRCol has multiple unique amino acidpolymorphisms centered in the NB domain and the linker re-gion between the NB and LRR domains (Figure 2E; seeSupplemental Figure 5A and Supplemental Data Set 2 online).The promoter sequences of the VICTR genes are highly con-served (see Supplemental Figure 5B and Supplemental DataSet 2 online).

VICTR Expression in DFPM-Insensitive AccessionsGenerates Col DFPM Responses

To further establish whether DFPM-induced primary root growthinhibition in Col-0 is dependent on polymorphisms present in thecoding region of VICTRCol, the VICTRCol gene expressed underthe control of its natural Col-0 promoter was introduced intoaccessions insensitive to DFPM: Ler, C24, and Bay-0. The in-troduction of VICTRCol was sufficient to generate the DFPMresponse in primary roots of Ler, C24, and Bay-0, demonstratingthat VICTRCol is the only missing component required for theDFPM-induced primary root growth arrest in these accessions(Figure 3A). As reported for other R proteins, ectopic 35S:VICTRCol expression in the Col-0 background caused stuntedplant growth (see Supplemental Figure 1D online).

VICTR Expression Is Increased in the Root MeristematicZone in a DFPM- and VICTR-Dependent Manner

Resistance genes are generally expressed ubiquitously and atvery low levels in unchallenged plants. Electronic Arabidopsis

gene expression resources, including AtGenExpress and eFPBrowser indicate that VICTR is expressed at a low level in manydifferent tissue types (Schmid et al., 2005; Winter et al., 2007).Interestingly, when treated with DFPM for 24 h, VICTR washighly induced (eightfold) in the Col-0 background (Figure 3B).Bay-0 and C24, which carry a polymorphic VICTR gene relativeto VICTRCol but are not sensitive to DFPM, did not exhibit en-hancement of VICTR expression in response to DFPM treatment(Figure 3B). More detailed analyses of VICTRCol promoterdriven-GUS (for b-glucuronidase) reporter and yellow fluores-cent protein (YFP) reporter lines showed dramatic DFPM-enhanced expression of the VICTR gene in the root meristematiczone (Figures 3C and 3D). These results suggest that the DFPM-induced root growth arrest may be caused by differential spatialupregulation of VICTR expression in the primary root tip region.Notably, induction of the VICTR gene by DFPM treatment

requires functional VICTRCol because DFPM could not inducethe VICTRCol promoter in the victr-1 mutant (Figure 3D, middlepanels). On the other hand, the promoter of VICTRBay, whosesequence is highly similar to that of VICTRCol (see SupplementalFigure 5B and Supplemental Data Set 2 online), activated YFPexpression by DFPM treatment when introduced into the Col-0wild type (Figure 3D, bottom panels). Altogether, these resultssuggest that functional VICTR mediates DFPM-induced positivefeedback regulation of VICTR expression and that amino acidpolymorphism(s) in VICTRCol are responsible for the VICTRphenotype. Consistent with this, Bay-0 expressing VICTRCol

under the VICTRCol promoter exhibited DFPM-mediated rootgrowth arrest (Figure 3A) and also increased VICTR expressionupon DFPM treatment (Figure 3B). Such positive feedbackregulation has been observed for R genes and is part of theresistance response induced by corresponding avirulentpathogens (Zhang and Gassmann, 2007).

VICTR Requires Effector-Triggered Immune SignalingComponents for the DFPM Response

The finding that VICTR encodes a TIR-NB-LRR family R-likeprotein and previous indications that DFPM induces defense-related gene expression (Kim et al., 2011) raise the hypothesisthat pathogen-triggered biotic stress signaling and/or salicylicacid (SA) signaling is responsible for generation of the VICTRroot growth arrest phenotype. To test this hypothesis and toidentify additional genetic components of the DFPM-mediatedsignaling pathway, pathogen mutants in Col-0 were analyzed todetermine whether they exhibit altered root growth responses toDFPM. Among the mutants analyzed, eds1-2 (Bartsch et al.,2006), eds1-23, and pad4-1 (Glazebrook et al., 1997) mutationsdisrupted the DFPM-induced root growth arrest, as did theeds1-2 pad4-1 double mutant (Figure 4A; see SupplementalFigure 4A online). No DFPM resistance was observed in eds5-1and eds16-1.EDS1 and PAD4 are also required for DFPM inhibition of ABA-

induced stomatal closure (Kim et al., 2011). Therefore, victr-1and victr-2 mutants were analyzed for the ability of DFPM toimpair ABA-induced stomatal closing in genotype blind assays.Both victr-1 and victr-2 plants showed impairment in DFPM in-hibition of ABA-induced stomatal closing (Figure 4B).

Natural Variation in TIR-NB-LRR Signaling 5181

Figure 3. VICTRCol Expression Is Sufficient for Generation of the DFPM-Induced Root Meristem Arrest in the DFPM-Resistant Ecotypes Ler, C24, andBay-0.

(A) Expression of the VICTRCol gene under the control of the VICTRCol promoter in Ler (left panel), C24 (middle), and Bay-0 (right) reproduced the Col-0phenotype in response to DFPM. Three independent transgenic lines are shown together with a control transgenic line transformed with empty vector.The black horizontal bars mark the starting position of roots when DFPM was applied.

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VICTR Subcellular Localization in TransientlyTransformed Cells

To investigate the subcellular localization of VICTR, YFP or GFPfluorophores were fused to either its N or C terminus. All con-structs showed that VICTRCol localized to the nucleus withlocalization also in the cytoplasm as indicated by faint fluores-cence in transient onion (Allium cepa) epidermis expressionassays (see Supplemental Figure 4D online). Arabidopsis TIR-NB-LRR proteins, such as RPS4 and RPS6, also displaynucleocytoplasmic distributions (Wirthmueller et al., 2007; Kimet al., 2009). In transient Agrobacterium tumefaciens–mediatedoverexpression and immunological detection of the VICTRconstructs in Nicotiana benthamiana plants, VICTR fusion pro-tein levels were low but detectable based on GFP fluorescenceand immunoblots (Figure 5). In N. benthamiana expressionanalyses, we coexpressed red fluorescent protein (RFP) con-structs, allowing us to clearly identify transformed cells, visualizenuclei, and perform colocalization analyses. The GFP-VICTRfusion protein was preferentially nuclear localized, and GFP-VICTR fluorescence merged with nuclear RFP (Figure 5A). Al-though >70% of cells showed RFP expression, GFP-VICTR wasdetected in only 15 to 20% of transformed cells. Immunoblotanalyses identified GFP-VICTR fusions at the expected size(;160 kD), suggesting that the detected fluorescence is not dueto either GFP alone or GFP fused to truncated VICTR (Figure 5B).

VICTR Resides in Protein Complexes with Nuclear EDS1and PAD4

The requirement for EDS1 and PAD4 in DFPM-dependent rootinhibition prompted us to investigate in vivo protein complexassociations of VICTR with these proteins. Recent studies haveshown that the TIR-NB-LRR resistance proteins RPS4 andRPS6 coreside in protein complexes with EDS1 (Bhattacharjeeet al., 2011; Heidrich et al., 2011). Pathogen effectors that mightsignal VICTR-dependent primary root growth arrest are presentlynot known and effectors that cause root growth arrest have not yetbeen determined. Such putative effectors may also redundantlyuse the tandem repeat homolog VICTL1. Identification of molec-ular interactors of VICTR is relevant toward understanding VICTRprotein functions. Bimolecular fluorescence complementation(BiFC), an efficient tool to study in vivo protein–protein interactions,can be used to identify molecular complexes of key innate im-munity components (Bhattacharjee et al., 2011). An inherent fea-ture of this assay is its irreversibility, allowing capture of transientinteractions. Confocal microscopy analysis of transiently ex-pressed BiFC fusion combinations of VICTR with PAD4 or EDS1 in

N. benthamiana cells suggested nuclear complexes of VICTR withthese two proteins (Figure 6). VICTR did not show associationswith the reciprocal BiFC vectors expressing GUS that also accu-mulated inside nuclei (see Supplemental Figure 6A online). Het-erodimers of EDS1-PAD4 display nucleocytoplasmic distributions(Feys et al., 2005). However, our BiFC results cannot distin-guish whether VICTR associates with EDS1-PAD4 heterodimer-including complexes or within protein complexes individually withEDS1 or PAD4 proteins, which warrants further investigations. Ourresults suggest cocomplex formation of a TIR-NB-LRR proteinwith the EDS1 signaling partner PAD4. This is in marked contrastwith the TIR-NB-LRR R proteins RPS4 and RPS6, which did notshow an interaction with PAD4 in transient expression assays(Bhattacharjee et al., 2011).We independently investigated the above results by per-

forming coimmunoprecipitation (co-IP) assays on transientlytransformed N. benthamiana cells (Figure 7A). N-terminallyhemagglutinin (HA) epitope-tagged VICTR (HA-VICTR)was coexpressed either with cMyc-tagged-EDS1, -PAD4 or-GUS. Because VICTR expression was enriched in the nu-cleus, we isolated purified nuclei from the infiltrated leaftissues and performed nuclear co-IP assays. Both EDS1 andPAD4, but not GUS, coimmunoprecipitated with VICTR(Figure 7A and 7C). These results validated the associationsof VICTR with EDS1 and PAD4 proteins in nuclei detected byBiFC (Figure 6).Using a mutated EDS1, eds1L262P, that does not hetero-

dimerize with PAD4 (Rietz et al., 2011), we tested whether VICTRassociates with EDS1-PAD4 heterodimers or individually withEDS1. As expected, the eds1L262P mutant showed a stronglydiminished interaction with PAD4 in transient N. benthamianaBiFC assays (Figure 7B). We then performed co-IP assayson transiently transformed N. benthamiana cells (Figure 7C). N-terminally HA epitope-tagged VICTR (HA-VICTR) was coex-pressed either with cMyc-tagged PAD4, -EDS1, -eds1L262P, or-GUS. Purified nuclei from the infiltrated leaf tissues were iso-lated and nuclear co-IP assays performed. EDS1 wild-type,eds1L262P, and PAD4, but not GUS, coimmunoprecipitated withVICTR (Figure 7C), supporting that VICTR can form complexeswith EDS1 and PAD4 inside nuclei.Senescence Associated Gene101 (SAG101) has also been

shown to form a protein complex with EDS1 (Feys et al., 2005;Rietz et al., 2011). As eds1-2 and pad4-1 loss-of-function mu-tants showed a strong DFPM insensitivity, we tested sag101-2loss-of-function mutants (Feys et al., 2005) for the DFPM-induced root growth arrest response. In contrast with eds1-2,pad4-1, and eds1-2 pad4-1, sag101-2 mutant plants behavedlike Col-0 wild-type controls (Figure 8C). Thus, we propose that

Figure 3. (continued).

(B) DFPM-sensitive Arabidopsis lines show induction of VICTR expression by DFPM treatment: five biological replicates for Col-0 and Bay-0 and threefor C24 and VICTRCol #2 (Bay-0).(C) VICTRCol promoter-driven GUS reporter lines in Col-0 background showed tissue-specific and DFPM-dependent induction of VICTR gene ex-pression. Five independent transgenic reporter lines are presented.(D) VICTRCol promoter-driven YFP reporter line confirms the root tip–specific expression of VICTR after DFPM treatment. No induction of the VICTRCol

promoter in victr-1 indicated that VICTRCol is required for the transcriptional DFPM induction of the VICTRCol promoter. The VICTRBay promoter droveYFP reporter expression similarly to the VICTRCol promoter. WT, the wild type. PI, propidium iodide.

Natural Variation in TIR-NB-LRR Signaling 5183

DFPM may trigger root growth inhibition by activating anSAG101-independent branch of EDS1-PAD4 signaling.

HSP90 Heat Shock Proteins and Their Cochaperones AreCrucial for DFPM Signaling

The HSP90 chaperones functionally stabilize and maintain Rproteins in a signaling-competent state, and their activity isdependent on at least two different cochaperones, RAR1 andSGT1B (Kadota et al., 2010). In root assays, the single mutantsrar1-21 (Tornero et al., 2002) and sgt1b/eta3 (Gray et al., 2003)exhibited DFPM insensitivity (Figure 8A). Since the Arabidopsisgenome contains four cytosolic HSP90 isoforms, with three lo-cated in close proximity within a 12-kb region on chromosome 5(Sangster et al., 2007), the effect of the HSP90 inhibitor gelda-namycin (GDA) on the DFPM response was analyzed. GDA in-hibits cancer cell proliferation by irreversibly binding to the Nterminus of HSP90 proteins (Stebbins et al., 1997). A concen-tration of 5 µM GDA was sufficient to completely abolish Col-0specific DFPM root growth inhibition (Figure 8B), consistent

Figure 4. DFPM-Induced Signal Transduction Requires Pathogen Sig-naling Components.

(A) Chemical signaling by VICTRCol is dependent on EDS1 and PAD4;thus, eds1-2 and pad4-1 mutants were insensitive to DFPM, in contrastwith eds5-1 and 35S:nahG. The black horizontal bars mark the startingposition of roots when DFPM was applied. Error bars show 6 SD (n = 3with 10 plants each).(B) VICTR functions in DFPM inhibition of ABA-induced stomatal closure.Guard cells of victr-1 and victr-2mutants remained sensitive to ABA evenin the presence of DFPM. n = 3 experiments with 42 to 50 stomata an-alyzed per condition. Error bars are SE. *P < 0.01.[See online article for color version of this figure.]

Figure 5. VICTR Localizes to the Nucleus in Plant Cells.

(A) Agrobacterium strains expressing GFP-VICTR fusions or GFP alonewere coinfiltrated with RFP-expressing strains in N. benthamiana leaves.Two days after infiltration, leaf sections were analyzed with a confocalmicroscope. The left and the middle panels show GFP and RFP fluo-rescence, respectively. The right panels show an overlap of GFP andRFP fluorescence. Arrowheads point out the nucleus. The assay wasrepeated with similar localizations. Bars = 20 mm.(B) Immunoblots (IB) detecting GFP-VICTR in N. benthamiana extractsand showing absence of free GFP or degradation products in GFP-VICTR sample.

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with the observed DFPM resistance of rar1-21 and sgt1b/eta3(Figure 8A). This suggests that HSP90 cooperates with RAR1and SGT1B to stabilize VICTR as a prerequisite for the DFPMroot growth response.

DFPM Response Does Not Require Jasmonic Acid orSA Signaling

The eds5-1 mutant defective in SA biosynthesis (Rogers andAusubel, 1997) and a SA-depleted 35S:nahG transgenic line(Reuber et al., 1998) were not affected in the DFPM-inducedroot growth response (Figure 4A). With the exception of theresistance signaling components described above, otherknown pathogen response loci were also not required for theDFPM-induced root growth arrest. These include the NB-LRRgenes RPS2, RPM1, and RPS4, immune signaling and re-sponse components NPR1, NPR3-NPR4, PAD3, and PMR4,and a pathogen-associated molecular pattern (PAMP) receptor,FLS2, as well as the jasmonic acid (JA) signaling componentsCOI1 and JAI1/JIN1 (Lorenzo et al., 2004; Zhang et al., 2006;Fonseca et al., 2009; Sheard et al., 2010; Fu et al., 2012) (Figure8C). Although application of exogenous SA also caused rootgrowth inhibition (see Supplemental Figure 7A online), detailed

inspection showed that the SA effect on root growth inhibitionis morphologically clearly distinct from the DFPM-induced rootgrowth arrest in several respects. SA blocks general growth ofArabidopsis seedlings (Zhang et al., 2003a), including rootelongation, lateral root initiation, and root hair formation (seeSupplemental Figure 7B online), in contrast with DFPM (e.g.,Figure 4A; see Supplemental Figure 2B online). Also, the SA-induced root growth inhibition is not accession specific be-cause the response to SA was equivalent in Col-0 and Ler.

DISCUSSION

We identified genetic natural variation in the VICTR locus thatproduces root growth arrest in response to the small moleculeDFPM. We report that DFPM perception and signal transductionrequires early components of the plant R gene resistance sig-naling network, including the TIR-NB-LRR protein VICTRCol,EDS1, and PAD4. This response may also involve VICTR sta-bilization by HSP90 together with RAR1 and SGT1B but oper-ates independently of major defense signaling components inthe JA and SA signal transduction pathways. VICTRCol is alsorequired for DFPM-induced inhibition of ABA-induced stomatalclosing, further showing that early TIR-NB-LRR signaling rapidlyinterferes with ABA signal transduction. We further establish intransient protein expression assays that VICTR resides withinnuclear protein complexes with EDS1 and PAD4. By contrast,the nucleocytoplasmic TIR-NB-LRR receptors RPS4 andRPS6 were found only in complexes with EDS1 and not PAD4(Bhattacharjee et al., 2011; Heidrich et al., 2011). Thus, differenttypes of TIR-NB-LRR receptors may operate in distinct resis-tance signaling complexes containing EDS1 and/or PAD4. Previ-ously, it was shown that the eds1L262P mutant, which does notinteract with PAD4 (Figure 7B), has defects in basal immunity tovirulent pathogens (Rietz et al., 2011). The retention of a co-IPsignal between VICTR and eds1L262P protein in transient ex-pression assays (Figure 7C) suggests that this mutation may notbe crucial for TIR-NB-LRR protein–EDS1 complex formation.A requirement for VICTR interactors EDS1 and PAD4 in the

DFPM-induced response and microarray analyses support thenotion that a plant effector-triggered immunity signaling path-way (Glazebrook, 2005; Wiermer et al., 2005; Kim et al., 2011)mediates the DFPM response (Figure 4A). Furthermore, the re-quirement of RAR1 and SGT1B and HSP90 chaperones sug-gests that VICTR steady state accumulation and possibly alsochaperone-mediated conformational changes are important forDFPM-triggered VICTR signaling (Shirasu, 2009) (Figures 8A and8B). However, the lack of requirement for other analyzed de-fense signaling elements, including key components of the SA-signaling and JA-signaling pathways (Lorenzo et al., 2004;Zhang et al., 2006; Fu et al., 2012), suggests that a SA/JA-independent disease signaling network participates in the DFPM-induced root growth arrest (Figures 4A and 8C). Since PIN1:GFPand PIN2:GFP expression was decreased after 18 h of DFPMtreatment, an involvement of auxin cannot be ruled out andwarrants further analysis. Auxin binding–specific fluorescencereporters (Brunoud et al., 2012) with a rapid readout may berequired to unequivocally resolve this question.

Figure 6. VICTR Associates with Nuclear Pool of EDS1 and PAD4.

Confocal analysis of N. benthamiana leaves agroinfiltrated with BiFCvector combinations of VICTR with EDS1 or PAD4. These vector com-binations use the N-terminal fragment of an enhanced version of YFP(nVenus) and the C-terminal fragment of CFP (cCFP), which when re-constituted gives yellow fluorescence (Hu and Kerppola, 2003). Locali-zation of indicated molecular complexes is shown by yellow fluorescence.Red fluorescence indicates free RFP. Panels show images with separateYFP, RFP, and an overlap of YFP + RFP fluorescence. Arrowheads in-dicate the nucleus. The experiment was repeated with identical results(n = 3). Bars = 20 mm.

Natural Variation in TIR-NB-LRR Signaling 5185

DFPM may activate TIR-NB-LRR–mediated signal transduc-tion by mimicking a pathogen effector (Belkhadir et al., 2004;Jones and Dangl, 2006; Caplan et al., 2008), analogous to theorganophosphate insecticide fenthion that triggers an immune-like response via the kinase Fen and the NB-LRR protein Prfin tomato (Solanum lycopersicum; Pedley and Martin, 2003).Activation of effector-triggered immunity can promote infectionby necrotrophic pathogens that feed off dead plant tissue(Glazebrook, 2005). Interestingly, certain necrotrophic fungalpathogens secrete small toxin molecules (such as victorin pro-duced by Cochliobolus victoriae), which are sensed by NB-LRR

proteins (Sweat and Wolpert, 2007; Lorang et al., 2012). Whenprimary roots are challenged by soil-borne pathogens, localizedVICTR responses may limit damage to the root and function inrestriction of primary root growth, thereby protecting roots fromfurther infection. Given the homology of VICTR and its tandemgene VICTL1, biological responses may be mediated re-dundantly via either of these two proteins. Chemical geneticsprovides a powerful approach for identification of redundantsignaling genes and their linked phenotypes by protein-specificselective activation, as in the case of the discovery of the re-dundant PYR1 ABA receptor (Park et al., 2009). Whether VICTR

Figure 7. VICTR Associates in Planta with EDS1, PAD4, and eds1L262P.

(A) Myc-tagged EDS1, PAD4, and GUS were transiently coexpressed with HA-VICTR in N. benthamiana leaves. Purified nuclear extracts were im-munoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies. Top panel shows the pull-down of PAD4 and EDS1, but not GUS, withVICTR. Bottom two panels show input fractions (n = 3).(B) eds1L262P does not strongly interact with PAD4 in BiFC assays. Reciprocal BiFC assays of PAD4 with wild-type EDS1 and eds1L262P were performedby Agrobacterium-mediated transient expression in N. benthamiana. Reconstituted YFP is indicated by yellow fluorescence. Bar values are indicated.(C) VICTR interacts with eds1L262P. Co-IP was performed as described above. Top panel is the co-IP of PAD4, EDS1, and eds1L262P, but not GUS, withVICTR. Bottom two panels show the input fractions. HA-VICTR was detected only after immunoprecipitation with anti-HA antibodies. Migration andsizes (in kilodaltons) of molecular mass standards are at the left of the respective immunoblot panels. Each experiment was performed twice with similarresults.

5186 The Plant Cell

Figure 8. The DFPM-Induced Signal Transduction Relies on HSP90 and Its Cochaperones but Does Not Require SAG101, SA, and JA SignalingComponents.

(A) Chemical signaling by VICTRCol requires functional HSP90 cochaperones RAR1 and SGT1B.(B) The DFPM signal was blocked by applying additional 5 mM of the HSP90 inhibitor GDA.(C) Other signaling and response components known to function in pathogen, SA, and JA signal transduction or other NB-LRR genes are not requiredfor the VICTR phenotype. All mutants were tested in Col-0 background. Error bars mean 6 SD. Note, some error bars are not visible due to very small SD(n = 3 with 10 plants each).[See online article for color version of this figure.]

Natural Variation in TIR-NB-LRR Signaling 5187

and VICTL1 function in concert in biologically induced rootgrowth arrest will require identification of natural effectors thatstimulate this root response in Arabidopsis.

DFPM was identified as a small molecule that rapidly inhibitsABA responses (Kim et al., 2011). DFPM interference with ABAsignal transduction also required PAD4, EDS1, RAR1, andSGT1B but not SA or JA signal transduction components (Kimet al., 2011). As shown in stomatal movement analyses, DFPMinhibition of ABA-induced stomatal closing is impaired in victrT-DNA insertion mutant stomata (Figure 4B), supporting theprevious model that DFPM mediates inhibition of ABA signaltransduction via early effector-triggered immune signaling (Kimet al., 2011). A requirement for VICTR in this response is con-sistent with data sets showing VICTR expression in leaves(Schmid et al., 2005; Winter et al., 2007).

TIR-NB-LRR genes mainly function in resistance againstpathogens. Retention of nonfunctional R gene alleles anda statistically significant increase in the occurrence of R genepolymorphisms is hypothesized to be an outcome of fitnesstradeoffs in evolving a large array of recognition specificities ina plant population (Tian et al., 2003; Weigel, 2012). An additionalconsequence of natural variants in NB-LRR genes can be en-vironmentally conditioned hybrid necrosis (Bomblies et al., 2007;Alcázar et al., 2009). TIR-NB-LRR–mediated root growth arresthas not been reported previously. Here, we show that DFPMinhibits root growth via VICTRCol in an EDS1/PAD4/RAR1/SGT1B/HSP90-dependent manner. These data suggest a directlink between R gene–related signaling and root growth arrest.DFPM causes a rapid root growth arrest along with a decreasedcell division rate in the primary root meristematic zone in Col-0.However, the quiescent center cells and cortical stem cellsappear unaffected after 24 h DFPM treatment in both resistantand sensitive genotypes, a time point at which initial root growthinhibition becomes visible. These findings and the DFPM-inducedVICTR promoter activity in wild-type plants suggest a role forVICTR protein levels in determining this interesting root meristem-localized EDS1- and PAD4-dependent response. As described fordelocalized or overexpressed R proteins (Zhang et al., 2003b; Yiand Richards, 2007; Kim et al., 2010), ectopic VICTR expressionalso leads to stunted growth phenotypes.

Identification of the small molecule DFPM as a trigger of aneffector-triggered immune-related signaling pathway that causesa strong visual and quantifiable primary root growth arrest canprovide a powerful tool for further genetic dissection of R gene–mediated signaling. This is demonstrated here by the identifica-tion of the VICTR gene and findings showing association of PAD4and EDS1 within VICTR protein complexes. Also, genetic variationin this small molecule response in Arabidopsis accessions isconsistent with the hypothesis that the repertoire of TIR-NB-LRRgenes provides activation mechanisms to diverse cues and herevia a root meristem-targeted response.

METHODS

Chemicals and Plant Materials

DFPM (ID 6015316) was isolated from screening of 9600 compounds inChemBridge’s DIVERSet E library. DFPM (ID 6015316), DFPM-1 (ID

5649754), DFPM-2 (ID 5872683), DFPM-3 (ID 6881095), DFPM-5 (ID6017927), and DFPM-7 (ID 5143713) were purchased from ChemBridge.Transgenic reporter lines were in the Col-0 background or were crossedinto the Col-0 background. Arabidopsis thaliana T-DNA lines were ob-tained from the ABRC (Ohio State University).

Arabidopsis ecotype set CS22660, victr-1 (Salk_123918), victr-2(Salk_084068), victr-3 (Salk_072727), victr-4 (Salk_122941), victr-5(Sail_394_F01), victl1-1 (Salk_097845), eds1-23 (Salk_057149), andCaroline Dean’s 100 Col 3 Ler RILs (CS1899) (Clarke et al., 1995;Singer et al., 2006) were obtained from the ABRC (Ohio State Uni-versity). The eds1-23 T-DNA mutant (Salk_057149) has a T-DNAinsertion in At3g48090, one of a tandem pair of EDS1 homologs inaccession Col-0. Another T-DNA insertion mutant (Salk_071051) inthe same gene, denoted eds1-22, substantially rescues the growthphenotype of bon1-1 constitutive resistance plants (Yang and Hua,2004). pDR5:GFP, pPIN1:PIN1:GFP, and pPIN2:PIN2:GFP werekindly provided by Yunde Zhao (University of California at San Diego).pSCR:GFP and pCyclinB1:GUS were kindly provided by Jeff Long(Salk Institute) and Jeff Harper (Univ. of Nevada), respectively. Mu-tants eds1-2 (Bartsch et al., 2006) and sag101-2 (Feys et al., 2005),pad4-1 and pmr4-1 (Nishimura et al., 2003), rar1-21 (Tornero et al.,2002), eta3/sgt1b (Gray et al., 2003), fls2 (Gómez-Gómez and Boller,2000), eds5-1 (Rogers and Ausubel, 1997) 35S:nahG line (Reuberet al., 1998), and npr3-2npr4-2 (Fu et al., 2012) were kindly providedby Jane Glazebrook (University of Minnesota), Jeff Dangl (Universityof North Carolina), William Gray (University of Minnesota), ShaunaSomerville (University of California at Berkeley), Sheng Yang He(University of Michigan), Mary Wildermuth (University of California atBerkeley), and Xin Li (University of British Columbia, Canada), re-spectively.

Root Growth Assays

Sterilized Arabidopsis seeds were germinated on general growth medium(1% Suc, 0.53 Murashige and Skoog [MS] salts, 0.05% MES, and 0.8%plant agar, pH 5.8) after 2 d of stratification and vertically grown for 10d before transfer to new plates containing the indicated chemicals. Aftertransfer onto new plates, the end of each root was marked as a startingpoint, and root growth arrest was monitored after 6 d. Root morphologyand GUS staining were examined by differential interference contrastmicroscopy (Leica DM5000B), and fluorescence reporter lines were an-alyzed by confocal microscopy (Nikon TE2000U) with propidium iodidestaining.

Confocal Microscopy

Col-0, PIN1:GFP, and PIN2:GFP plants were grown vertically in half-strengthMSmedium containing 1%Suc for 5 d and transferred to 10 mMDFPM half-strength MS medium. At the indicated time, primary rootswere stained with propidium iodide and observed using a Zeiss LSM 710confocal microscope with spectral settings for excitation at 488 nm/emission at 493 to 555 nm for the GFP signal and excitation 514 nm andemission at 596 to 719 nm for propidium iodide. A constant gain valuewas used for each genotype. For root meristem cell quantification, Col-0,victr-1, and victr-2 plants were prepared as described above. Meristemcells in the division zone were counted using Image J (Schneider et al.,2012). A single file of cortex cells was analyzed in all roots. The divisionzone was defined as the region made up of isodiametric cells between thequiescent center up to the cell, which was twice the length of the cellimmediately before it (González-García et al., 2011). analysis of varianceand Tukey test statistical analyses were conducted using the Origin-Proversion 8.6 package. Ten seedlings per genotype and treatment wereanalyzed.

5188 The Plant Cell

QTL Analyses and VICTR Cloning

For initial mapping and QTL analyses, the DFPM-induced VICTR phe-notype was scored on the 100 Col 3 Ler RILs. QTL analyses wereperformed using QTL package (version 1.11-12) that uses R software(version 2.81) based on the QTL package manual (Broman et al., 2003).Parameters used for LOD estimation were step = 1 and error probability =0.01. Haldane map function was used as a mapping algorithm. Genotypinginformation of each Col3 Ler RILs was retrieved from the microarray-basedphysical genomic mapping data set (Singer et al., 2006).

For marker-based cloning of the VICTRCol locus, the recessive Ler rootdevelopment phenotype was screened from a mapping population of;2000F2 plants of Col crosses to Ler. The VICTRCol locus was mapped to an ;5-centimorgan interval on the lower arm of chromosome 5 based on linkage toPCR-basedmarkers 18.1Mb (59-GTCGTAGAAGTAGCAGCTTGTACGAAGT-39/59-TCTCAATCCAATGTTCAGGTGACACACT-39) and 19.1 Mb (59-GAA-TCTCTAACGATGCTAACACAAGGA-39/59-CTGCAATCACAGTTTAAGTCAT-GAACT-39). Within the 100-kb region defined bymarkers 18.86 and 18.95Mb,21 T-DNA insertion lines targeting 14 genes among 18 predicted genes(At5g46490 to At5g46650) were analyzed for the root VICTR phenotype.

Deletion of VICTR in certain ecotypes (see Supplemental Figure 4 online)was examined by PCR amplification of genomic DNA templates usingLA Taq and Ex Taq polymerases (Takara Bio). Control primers are59-ATGGTTGCGATACCCAATATTAAGA-39 and 59-CTAAAGGATCT-CTCCTTCCTCAAGATCGT-39. Internal primers are 59-CGGGTAGA-AATATTGTACGCTCACAGT-39 and 59-CCAAATGGTTGAGAGTCAG-ATGTTC-39. Note that internal primers anneal to both VICTR andVICTL1. External primers are 59-GGACGCTTTGTTTCCAGCTTGTG-GGGCATTAAATCGAG-39 and 59-CACTTCGTAGGGCGTTGGGGCA-GACATTGCCTTCTC-39.

Transgenic Studies

To generate VICTR promoter-driven reporter lines, a 2.5-kb fragment from thestart codon of Col-0 and Bay-0 was PCR amplified from genomic DNA usingprimers (59-GGGGACAAGTTTGTACAAAAAAGCAGGCTGAGAACCAGTA-CACAATGGCAGGTAAG-39/59-GGGGACCACTTTGTACAAGAAAGCTGGG-TAAGAAGCCATAGAGAGAGTTAGAAGGAGGA-39) and cloned into pBGGUSand pHGY (vector plasmids were kindly provided by Taku Demura fromRIKEN) (Kubo et al., 2005). The VICTRCol gene was PCR amplified usingprimers 59-GGGGACAAGTTTGTACAAAAAAGCAGGCTGCCCTGTGGTA-TCTGGTAATCAAAATTCTGT-39/59-GGGGACCACTTTGTACAAGAAAGC-TGGGTGGCTACAAAGCAAGTCAGTCGTTCGTTG-39. The VICTRCol codingregion for the 35S promoter driven transgenic line was PCR amplified usingprimers 59-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCCTCCTTCTAA-CTCTCTCTATGGCTTC-39/59-GGGGACCACTTTGTACAAGAAAGCTGGG-TCTCATATGAAGTGGTGTAGCTGCAAAAG-39. For recapitulation of theCol-0 VICTR phenotype in Ler and C24, the VICTRCol gene including thepromoter and coding region was inserted into pHGY. For generation of35S promoter–driven VICTRCol transgenic lines, the VICTRCol codingregion, including introns, was cloned into pH35GS. Transformed lines withempty vector were used as controls for phenotypic analyses.

For subcellular localization analysis in onion (Allium cepa) epidermalcells (see Supplemental Figure 4D online), full-length VICTRCol cDNA wasinserted into pH35GY and pH35YG to construct plasmids encodingVICTR-YFP and YFP-VICTR, respectively. Transient expression by biol-istic bombardment (using PDS-1000/He; Bio-Rad) was performed inonion epidermal cells.

Localization and Immunodetection of VICTR in Nicotianabenthamiana Cells

VICTR cDNA in the entry vector (pENTR-TOPO-VICTR) was transferredvia Clonase LR recombination system (Invitrogen) into the Gateway-

compatible destination vector pMDC43 (Curtis and Grossniklaus, 2003),resulting in a GFP-VICTR expression vector. The RFP vector used incolocalization analysis was described earlier (Bhattacharjee et al., 2011).N. benthamiana leaves were infiltrated with the indicated Agrobacteriumtumefaciens strains at an OD600 of 0.3 for each strain. Two days afterinfiltration, tissue sections from the infiltrated patches were analyzed by atwo-photon equipped Zeiss confocal microscope. Three 7-mm-diameterleaf punches were excised from the infiltrated tissues and ground in 100mL of 8 M urea and subjected to immunoblotting with anti-GFP antibodies(Sigma-Aldrich).

BiFC Assays

The pENTR-TOPO-VICTR clone was recombined into the Gateway-compatible BiFC vectors pMDC-nVenus or pMDC-cCFP (Bhattacharjeeet al., 2011) using the Clonase LR recombination system. BiFC vectors forEDS1, PAD4, and GUS expression were used as in previous studies(Bhattacharjee et al., 2011). Agroinfiltration and confocal microscopyanalysis were performed as indicated earlier. For fusion protein detection,three 7-mm-diameter leaf discs were excised from the infiltrated patches,ground in 8 M urea and analyzed by immunoblotting using anti-GFPantibodies (Santa Cruz Biotechnology).

Coimmunoprecipitation Analyses

HA-tagged VICTR was generated using pENTR-TOPO-VICTR and thedestination vector HA-pBA (Kim et al., 2010). Myc-tagged EDS1, PAD4,and GUS have been used before (Bhattacharjee et al., 2011). Theeds1L262P mutant described by Rietz et al. (2011) was generated byoverlap PCR using the primers 59-TTCCCTGAGCTAAGTCCTTATA-39and 59-GCTCAGGGAAACTAGAAAGGGT-39 and replacing the wild-typefragment in the entry clone of EDS1. Myc-eds1L262P was generated byClonase recombining the entry clone into the Myc-pBA binary vector.Agrobacterium-infiltrated N. benthamiana leaves were processed for nu-clear extracts as described by Palma et al. (2007). The resulting nuclearlysates were centrifuged at 14,000g for 10 min to remove nuclear debris.Immunoprecipitation was performed as published before (Bhattacharjeeet al., 2011). The immunoprecipitates and the input fractions were analyzedwith anti-HA (Roche) or anti-Myc (Santa Cruz Biotechnology) antibodies.

Quantitative Real-Time RT-PCR

Col-0, C24, Bay-0, and one line of Bay-0 expressing VICTRCol-0 weregrown in half-strength MS media for 14 d and were transferred to half-strength MS containing 10 mMDFPM. After 24 h of DFPM treatment, totalRNA was extracted, and real-time RT-PCR was performed to quantify thetranscript levels of VICTR. Primers were designed with QuantPrime(Arvidsson et al., 2008): VICTR-qRT2-F, 59-AGAGACCGGTTCATCAG-CAGAG-39;VICTR-qRT2-R, 59-CCATATTGCCTTCTTCGGCTTGAG-39.Amplified samples were normalized against PDF2 levels: PDF2_F, 59-TAACGTGGCCAAAATGATGC-39; PDF2_R, 59-GTTCTCCACAACCGCT-TGGT-39.

Stomatal aperture measurements were performed as described before(Kim et al., 2011; Hubbard et al., 2012). Student’s t tests were performedfor n = 3 independent experiments with 42 to 50 stomata analyzed foreach condition.

Accession Numbers

Sequence data from this article can be found in the ArabidopsisGenome Initiative database under the following accession numbers:VICTR, At5g46520; VICTL1, At5g46510; EDS1, At3g48090; and PAD4,AT3G52430.

Natural Variation in TIR-NB-LRR Signaling 5189

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Functional Characterization of ModifiedDFPM Structures and 35S:VICTRCol Ectopic Expression CausedStunted Growth.

Supplemental Figure 2. Gene Expression and MorphologicalChanges of DFPM-Treated Seedlings.

Supplemental Figure 3. DFPM Reduces the Number of MeristemCells in the Wild Type, but Not in victr Mutant Alleles.

Supplemental Figure 4. Characterization of eds1-23 T-DNA InsertionLine and victl1-1, Subcellular Localization of VICTR, and VICTRDeletion in Other Accessions.

Supplemental Figure 5. VICTR Gene Sequence Comparison of Col-0with Bay-0 and Kin-0.

Supplemental Figure 6. VICTR Does Not Associate with GUS.

Supplemental Figure 7. Root Growth Inhibition by Salicylic AcidDiffers from the DFPM-Induced Primary Root Growth Arrest Re-sponse.

Supplemental Table 1. List of Arabidopsis Accessions That Did NotProduce the Root Developmental Arrest in Response to DFPMTreatment.

Supplemental Table 2. DFPM-Induced Root Growth Arrest VICTRPhenotype in 100 Col 3 Ler Recombinant Inbred Lines.

Supplemental Data Set 1. Text File of the Alignment Used toGenerate the Phylogenetic Tree Shown in Supplemental Figure 4B.

Supplemental Data Set 2. Text File of the Alignment in SupplementalFigure 5.

ACKNOWLEDGMENTS

We thank Chris Somerville (University of California at Berkeley) forproviding access to the chemical library of 9600 compounds, Jeff Danglfor providing rar1-21 seeds, and Amber Ries [University of California atSan Diego (UCSD)] for independent blinded stomatal movement analyses.We thank Rosangela Sozzani (University of Pavia), Yunde Zhao,Sangho Jeong, Aurelien Boisson-Dernier, Noriyuki Nishimura andCarolyn Rasmussen at UCSD for comments on the article and helpfuldiscussions. This research was supported by the National Institutes ofHealth (R01GM060396; J.I.S.), the National Science Foundation(MCB0918220, J.I.S.; IOS1121114, W.G.), and the Max-Planck Societyand Deutsche Forschungsgemeinschaft ’SFB 670’ (J.E.P) grants.Root growth analyses were in part funded by a grant from theDivision of Chemical Sciences, Geosciences, and Biosciences, Officeof Basic Energy Sciences of the U.S. Department of Energy (DE-FG02-03ER15449; J.I.S.) and in part by the National Research Foundation ofKorea funded by the Ministry of Education, Science and Technology(2012-0002781 and 2012-041653) to T.-H.K. H.-H.K. was funded by theHuman Frontier Science Program and the Alexander von HumboldtFoundation.

AUTHOR CONTRIBUTIONS

T.-H.K. and J.I.S. designed the experiments. T.-H.K. performed most ofthe experiments. S.B., H.-H.K., W.G., and J.I.S. designed BiFC and co-IPexperiments. S.B. and H.-H.K prepared constructs and S.B. conductedBiFC and co-IP experiments. H.-H.K. worked on GDA/DFPM interference

and distributed material. A.L. conducted stomatal movement analyses.T.-H.K., F.H., H.-H.K., W.G., J.E.P., and J.I.S. analyzed the data. C.E.analyzed meristematic cell division, J.P. contributed quantitative PCR,pPIN∷PIN:GFP results, and repeated pDR5:GFP data. S.B., H.-H.K.,W.G., and J.E.P. contributed to the eds1L262P experiment. T.H. assistedwith the VICTR cloning. T.-H.K., H.-H.K., and J.I.S. wrote the article withinput from other coauthors.

Received November 8, 2012; revised November 8, 2012; acceptedDecember 13, 2012; published December 28, 2012.

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5192 The Plant Cell

DOI 10.1105/tpc.112.107235; originally published online December 28, 2012; 2012;24;5177-5192Plant Cell

Engineer, Amy Liu, Tracy Ha, Jane E. Parker, Walter Gassmann and Julian I. SchroederTae-Houn Kim, Hans-Henning Kunz, Saikat Bhattacharjee, Felix Hauser, Jiyoung Park, Cawas

ArabidopsisArrest via EDS1- and PAD4-Complexed R Protein VICTR in Induced TIR-NB-LRR Signaling Induces Root Growth−Natural Variation in Small Molecule

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